Adenosine Diphosphate Degradation Research Articles

Mechanism of Graft Damage Caused by NTPDase1-activated Macrophages in Acute Antibody-Mediated Rejection

ObjectivesTo investigate the effect and mechanism of macrophage activation and graft damage caused by nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) in acute antibody-mediated rejection (AMR). MethodsAcute AMR was induced in different skin-grafted nude mouse models with wild-type NTPDase1 expression, transgene-enhanced NTPDase1 expression, or NTPDase1 gene knockout. Several methods (eg, real-time fluorescence quantitative polymerase chain reaction, high-performance liquid chromatography [HPLC], immunofluorescence, flow cytometry, and luciferin/luciferase assays) were used to study (at the histologic and molecular levels) the extracellular adenosine diphosphate (ADP) concentration, macrophage proliferation, major histocompatibility complex (MHC) class II antigen expression on the surface of macrophages, B-cell activating factor (BAFF) expression in the peripheral blood serum, and the total number of SmIg-positive B cells during acute AMR. The relative activity of NTPDase1 in B cells and epithelial cells, pathologic changes, and the incidence of positive C4d deposition around the capillaries of skin grafts on the different nude mice were studied. ResultsMacrophages proliferated significantly when acute AMR occurred. The higher the NTPDase1 expression level, the lower the extracellular ADP concentration, the expression of MHC class II antigens on the surface of macrophages, the expression of BAFF in the peripheral blood serum, and the total number of SmIg-positive B cells, indicating negative correlations. The relative activity of NTPDase1 in B cells and epithelial cells of the skin graft was different among the different mice. The higher the NTPDase1 expression level, the lower the degree of pathologic damage to the skin graft. ConclusionsImbalance in extracellular ADP degradation by NTPDase1 may promote macrophage activation, and activated macrophages may be an important cause of graft damage.

ITRAQ-based proteomic analysis of human umbilical vein endothelial cells with platelet endothelial aggregation receptor-1 knockdown.

The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y12 , mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.

Mechanism of Nucleoside Triphosphate Diphosphohydrolase-1–Associated Imbalance in Adenosine Diphosphate Degradation, B-Cell Activation, and Related Injury During Acute Antibody-Mediated Rejection

ObjectiveThe objective of this study was to investigate the effect of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) during acute antibody-mediated rejection (AMR). MethodsNTPDase1 overexpression, NTPDase1 knockout, and wild-type nude mice skin graft models were used to induce acute AMR. NTPDase1 expression in B cells, NTPDase1 messenger RNA expression in skin grafts, extracellular adenosine diphosphate (ADP) concentration, B-cell volume and surface antigens expression, average platelet transport rate, and ultrastructure and apoptosis of skin graft cells were investigated. ResultsDuring acute AMR in nude mice, higher NTPDase1 expression caused lower extracellular ADP concentration, smaller increase in B-cell volume, and major histocompatibility complex II surface antigen expression, suggesting a negative correlation between them; higher NTPDase1 expression also caused slower average platelet transport rate and less severe skin graft injury, suggesting a negative correlation between them. Pretreatment with high-dose exogenous NTPDase1 inhibited platelet activation and protected skin grafts, but it resulted in prolonged bleeding time (by 51.4%) and prolonged coagulation time (by 44.1%). ConclusionAn NTPDase1-associated imbalance in extracellular ADP degradation may contribute to B-cell activation, platelet activation, and more severe skin graft injury in nude mice. Pretreatment with high-dose exogenous NTPDase1 effectively protected skin grafts in nude mice at 1 week, but it increased the risk of bleeding.

Underlying mechanism and specific prevention of hemolysis-induced platelet activation

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, “spontaneous” platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.

Innate immunity, purinergic system, and liver regeneration: A trip in complexity

Innate immunity, purinergic system, and liver regeneration: A trip in complexity

In Vitro Verification of Antithrombotic Effect of Recombinant Soluble Nucleotide Triphosphate Diphosphohydrolase 1

Ecto-ADPase (NTPDase1 or CD39) has been identified on endothelial cells and found to be antithrombogenic, with actions resulting from degradation of adenosine diphosphate (ADP), a thrombogenic molecule secreted by activated platelets at sites of vascular injury. Reasoning that the ADPase activity of CD39 might provide clinical use as an antithrombotic agent, the authors investigate the comparative ability of the agent to inhibit platelet and fibrin deposition. With use of an in vitro perfusion system, fresh, heparinized human blood was passed over expanded polytetrafluoroethylene grafts at hemodynamic conditions similar to those observed in the human arterial circulation. Three different concentrations of CD39 (30 mcg/mL, 100 mcg/mL, and 300 mcg/mL) were compared with abciximab (4 mcg/mL) and heparin controls. The deposition of radiolabeled platelets and fibrinogen was measured in five perfusions for each treatment group. The addition of soluble CD39 to heparinized blood inhibited platelet deposition to an extent greater than that of heparin alone (P =.04). Effects were similar to those observed with abciximab. The addition of CD39 to heparinized blood did not result in augmentation of fibrin inhibition beyond that observed with heparin alone. The fibrin inhibitory effects of CD39 appeared to be less significant than the fibrin inhibition observed with abciximab, but this difference did not attain statistical significance. These results suggest that recombinant CD39 holds potential as antithrombotic agent, with the potential to achieve antiplatelet effects similar to that observed with abciximab.

The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 μl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca 2+/Mg 2+-dependent and Ca 2+/Mg 2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0–2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 μM NaN 3) and non-specific alkaline phosphatase (β-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN 3, suramin, ATPγS, La 3+ and CTP, the hydrolysis of ADP by β,γ-imidoATP, and AMP degradation by α,β-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5′-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated K m values of 2282 μM, 6619 μM and 881 μM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.

Properties of ADP-degrading activity of human placental syncytiotrophoblast brush border membrane vesicles

The enzymatic properties of ADP (adenosine diphosphate) degradation in human placental syncytiotrophoblast brush border membrane vesicles (BBMV) were explored and the following results were obtained. BBMV had high ADP degrading activity compared to homogenate of placental villi. ADP degrading activity of BBMV; 1.05 +/- 0.05 mumol/mg protein/min placental villi was 21 times higher than that of homogenate of placental villi. Hydrolysis of ADP by BBMV follows Michaelis-Menten saturation kinetics with an apparent Km of 10.9 +/- 0.8 microM and Vmax of 2.10 +/- 0.17 mumol/mg protein/min. The enzyme has a divalent cation requirement. EDTA (2 mM) was found to abolish ADP degrading activity but this could be restored by the addition of either magnesium or calcium ions. Maximum enzyme activity of ADP degradation in BBMV was observed at a pH close to 8.0. The enzyme was insensitive to vanadate, levamisole, oligomycin, ouabain and N-ethylmaleimide (NEM), omeprazole and adenosine (5') pentaphospho (5') adenosine.

Demonstration of antithrombotic activity of glomerular adenosine diphosphatase [published erratum appears in Blood 1991 Oct 15;78(8):2163

We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.

Demonstration of Antithrombotic Activity of Glomerular Adenenosine Diphosphatase

Abstract We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.

Effects of aspirin and dipyridamole on the degradation of adenosine diphosphate by cultured cells derived from bovine pulmonary artery.

To improve understanding of the mechanisms by which ADP is degraded during passage through the pulmonary vascular bed, we examined cultured endothelial and smooth muscle cells of bovine pulmonmary artery for their abilities to metabolize [8-14C]ADP. ADP is rapidly converted to AMP and then to adenosine, hypoxanthine, and inosine. Inosine is the major metabolite produced by endothelial cells. Radioactivity (5-10%) is accumulated intracellularly primarily as ATP. Medium containing 50 micro M ADP incubated with endothelial cells rapidly loses its ability to aggregate platelets and becomes antiaggregatory under conditions in which prostacyclin is absent. The antiaggregatory activity is probably the result of accumulated adenosine. 10 micro M dipyridamole inhibits cellular uptake of radioactivity by greater than 90%, and inosine in the medium is largely replaced by adenosine. This is accompanied by increased anti-aggregatory activity of conditioned medium, which can be matched by authentic adenosine at the same concentration. 1 mM aspirin had no effect on the metabolism of ADP by endothelial cells. Our results suggest: (a) Metabolism of ADP during passage through the lung is mainly the result of endothelial ADPase. (b) ADP released from aggregating platelets can be converted to the antiaggregatory substance, adenosine. Dipyridamole may exert some of its antithrombotic actions by preventing the intracellular uptake of adenosine, thereby increasing its concentration near the site of thrombus formation. (c) The ability of the vessel wall to degrade ADP should not be compromised by the use of aspirin as an antithrombotic drug. (d) Endothelium may retain some of its antithrombogenicity when prostacyclin generation is impaired.

The Inhibition of Platelet Aggregation by an Aorta Intima Extract

SummaryThe platelet aggregating activity of extracts of different layers of the arterial wall was compared to that of Achilles tendon. Arterial media and tendon extracts, adjusted to equivalent protein content as an index of concentration, aggregated platelets to the same extent but an arterial intima extract did not aggregate platelets. Platelet aggregation induced by collagen could be inhibited by mixing with intima extract, but only to a maximum of about 80%. Pre-mixing adenosine diphosphate (ADP) with intima extracts diminished the platelet aggregation activity of the ADP. Depending on the relationship between ADP and intima extract concentrations aggregating activity could either be completely inhibited or inhibition abolished. Incubation of ADP with intima extract and subsequent separation of degradation products by paper chromatography, demonstrated a time-dependent breakdown of ADP with AMP, adenosine, inosine and hypoxanthine as metabolic products; ADP removal was complete. Collagen, thrombin and adrenaline aggregate platelets mainly by endogenous ADP of the release reaction. Results of experiments comparing inhibition of aggregation caused by premixing aggregating agent with intima extract, before exposure to platelets, and the sequential addition of first the intima extract and then aggregating agent to platelets, suggest that the inhibitory effect of intima extract results from ADP breakdown. It is suggested that this ADP degradation by intima extract may play a protective role in vivo by limiting the size of platelet aggregates forming at the site of minimal “wear and tear” vascular trauma.

Investigation on Adenosine Diphosphate (ADP) Induced Platelet Adhesiveness in Vitro Part III. The Inactivation of ADP in Plasma

SummaryThe results of investigations on the ADP inactivating system in platelet poor plasma are given. The activity of the system is maximal at about 37° C. and is irreversibly destroyed by heating for 15 minutes at 58° C. The system is non dialysable and is inactivated by KCN, cysteine, 2.3-dimercaptopropanole (BAL) and penicillamine at concentrations between 2 · 10∼3 and 5 · 10−3M.The ADP inactivating ability of platelet poor plasma from normals, patients with thyrotoxicosis, diabetes mellitus, hypercholesterolemia and some patients with bleeding disorders has been estimated. The inactivation by plasma from patients with thyrotoxicosis is markedly increased as compared with the other groups. A possible relationship between the low frequency of thromboembolism and the increased ADP degradation in these patients is discussed.